Abstract. Nature is the ultimate synthetic chemist, and metalloenzymes the catalysts of choice. To achieve their unprecedented functional diversity, metalloenzymes modulate the structural and electronic properties of the protein environment in which the reaction proceeds, thus enabling difficult transformations that are often challenging for synthetic chemists. Although enormous progress has been made in the study of structural and mechanistic enzymology, the dynamics of these reactions remain poorly understood. This proposal uses innovative methods to characterize the concerted atomic and electronic variations that facilitate catalysis in two medicinally-relevant classes of iron-containing metalloenzymes. Experiments will be rationally designed based on known mechanistic behavior to!visualize otherwise transient catalytic intermediates both in solution and in crystallo. The first specific aim focuses on the mode of substrate binding and ferryl-heme formation in the immunosuppressive human enzyme indoleamine 2,3-dioxygenase. This project will be completed during the K99 funding period and will involve crystallographic characterization of both the reactant complex and an enzymatically-generated ferryl species. Intermediates will be stabilized using substrate/cofactor mimics, site- directed mutants, or freeze-trapping methods exploiting the pH dependence of the reaction. During the independent phase, the second aim will use a similar approach applied to study the relatively uncharacterized class of cobalamin-dependent radical S-adenosylmethionine methyltransferases, involved in the biosynthesis of potent antibiotics. Although intermediate state models are desirable, any structure would provide unique insight into the mechanism of this class as none have been published to-date. The third and final aim, to develop and apply a laser pump/X-ray probe setup for the simultaneous collection of X-ray crystallographic and spectroscopic real time data, will be pursued concurrently. Princeton University provides the ideal environment in which to initiate pursuit of these goals, with excellent facilities and access to the world?s leading experts in bioinorganic chemistry. These resources will be complemented by my co-mentor at the Pennsylvania State University. Having received a formal education in physics, my short-term goals are to acquire wet lab skills necessary to generate, characterize and troubleshoot my own samples. I will be trained in protein expression and purification as well as UV-vis, EPR and sophisticated crystallographic characterization of metalloenzymes. During the mentored phase, I will attend a formal course in heterologous expression and purification of proteins, as well as a number of other workshops/conferences designed to increase my exposure to these techniques and learn the management skills required to be a successful PI. The expertise I acquire in the K99 period will be necessary to study more complex systems and develop laser pump/X-ray probe time-resolved methods for the study of metalloenzymes during the independent phase. In the long-term, I plan to lead a multidisciplinary research program at the interface of protein biochemistry and X-ray science.